Muscle and Bone Defects in Metastatic Disease.

PURPOSE OF REVIEW: The present review addresses most recently identified mechanisms implicated in metastasis-induced bone resorption and muscle-wasting syndrome, known as cachexia. RECENT FINDINGS: Metastatic disease in bone and soft tissues is often associated with skeletal muscle defects. Recent studies have identified a number of secreted molecules and extracellular vesicles that contribute to cancer cell growth and metastasis leading to bone destruction and muscle atrophy. In addition, alterations in muscle microenvironment including dysfunctions in hepatic and mitochondrial metabolism have been implicated in cancer-induced regeneration defect and muscle loss. Moreover, we review novel in vitro and animal models including promising new drug candidates for bone metastases and cancer cachexia. Preservation of bone health could be highly beneficial for maintaining muscle mass and function. Therefore, a better understanding of molecular pathways implicated in bone and muscle crosstalk in metastatic disease may provide new insights and identify new strategies to improve current anticancer therapeutics.

Iron overload in aging Bmp6­/­ mice induces exocrine pancreatic injury and fibrosis due to acinar cell loss.

The relationship between hemochromatosis and diabetes has been well established, as excessive iron deposition has been reported to result in impaired function of the endocrine and exocrine pancreas. Therefore, the objective of the present study was to analyze the effects of iron accumulation on the pancreata and glucose homeostasis in a bone morphogenetic protein 6­knockout (Bmp6­/­) mouse model of hemochromatosis. The sera and pancreatic tissues of wild­type (WT) and Bmp6­/­ mice (age, 3 and 10 months) were subjected to biochemical and histological analyses. In addition, 18F­fluorodeoxyglucose biodistribution was evaluated in the liver, muscle, heart, kidney and adipose tissue of both animal groups. The results demonstrated that 3­month­old Bmp6­/­ mice exhibited iron accumulation preferentially in the exocrine pancreas, with no signs of pancreatic injury or fibrosis. No changes were observed in the glucose metabolism, as pancreatic islet diameter, insulin and glucagon secretion, blood glucose levels and glucose uptake in the liver, muscle and adipose tissue remained comparable with those in the WT mice. Aging Bmp6­/­ mice presented with progressive iron deposits in the exocrine pancreas, leading to pancreatic degeneration and injury that was characterized by acinar atrophy, fibrosis and the infiltration of inflammatory cells. However, the aging mice exhibited unaltered blood glucose levels and islet structure, normal insulin secretion and moderately increased α­cell mass compared with those in the age­matched WT mice. Additionally, iron overload and pancreatic damage were not observed in the aging WT mice. These results supported a pathogenic role of iron overload in aging Bmp6­/­ mice leading to iron­induced exocrine pancreatic deficiency, whereas the endocrine pancreas retained normal function.

A cell-based evaluation of a non-essential amino acid formulation as a non-bioactive control for activation and stimulation of muscle protein synthesis using ex vivo human serum.

PURPOSE: The purpose of this study was to compare the effect of treating skeletal muscle cells with media conditioned by postprandial ex vivo human serum fed with either isonitrogenous Non-Essential Amino Acid (NEAA) or a whey protein hydrolysate (WPH) on stimulating Muscle Protein Synthesis (MPS) in C2C12 skeletal muscle cells. METHODS: Blood was taken from six young healthy males following overnight fast (fasted) and 60 min postprandial (fed) ingestion of either WPH or NEAA (0.33 g.kg-1 Body Mass). C2C12 myotubes were treated with media conditioned by ex vivo human serum (20%) for 4 h. Activation of MPS signalling (phosphorylation of mTOR, P70S6K and 4E-BP1) were determined in vitro by Western Blot and subsequent MPS were determined in vitro by Western Blot and surface sensing of translation technique (SUnSET) techniques, respectively. RESULTS: Media conditioned by NEAA fed serum had no effect on protein signalling or MPS compared to fasted, whereas media conditioned by WPH fed serum significantly increased mTOR (Ser2448), P70S6K and 4E-BP1 phosphorylation (p<0.01, p<0.05) compared to fasted serum. Furthermore, the effect of media conditioned by WPH fed serum on protein signalling and MPS was significantly increased (p<0.01, p<0.05) compared to NEAA fed serum. CONCLUSION: In summary, media conditioned by NEAA fed serum did not result in activation of MPS. Therefore, these in vitro findings suggest the use of isonitrogenous NEAA acts as an effective control for comparing bioactivity of different proteins on activation of MPS.

Recombinant Human Bone Morphogenetic Protein 6 Delivered Within Autologous Blood Coagulum Restores Critical Size Segmental Defects of Ulna in Rabbits.

BMP2 and BMP7, which use bovine Achilles tendon-derived absorbable collagen sponge and bovine bone collagen as scaffold, respectively, have been approved as bone graft substitutes for orthopedic and dental indications. Here, we describe an osteoinductive autologous bone graft substitute (ABGS) that contains recombinant human BMP6 (rhBMP6) dispersed within autologous blood coagulum (ABC) scaffold. The ABGS is created as an injectable or implantable coagulum gel with rhBMP6 binding tightly to plasma proteins within fibrin meshwork, as examined by dot-blot assays, and is released slowly as an intact protein over 6 to 8 days, as assessed by ELISA. The biological activity of ABGS was examined in vivo in rats (Rattus norvegicus) and rabbits (Oryctolagus cuniculus). In a rat subcutaneous implant assay, ABGS induced endochondral bone formation, as observed by histology and micro-CT analyses. In the rabbit ulna segmental defect model, a reproducible and robust bone formation with complete bridging and restoration of the defect was observed, which is dose dependent, as determined by radiographs, micro-CT, and histological analyses. In ABGS, ABC scaffold provides a permissive environment for bone induction and contributes to the use of lower doses of rhBMP6 compared with BMP7 in bovine bone collagen as scaffold. The newly formed bone undergoes remodeling and establishes cortices uniformly that is restricted to implant site by bridging with host bone. In summary, ABC carrier containing rhBMP6 may serve as an osteoinductive autologous bone graft substitute for several orthopedic applications that include delayed and nonunion fractures, anterior and posterior lumbar interbody fusion, trauma, and nonunions associated with neurofibromatosis type I.

A novel role of bone morphogenetic protein 6 (BMP6) in glucose homeostasis.

AIMS: Bone morphogenetic proteins (BMPs) are involved in the development and homeostasis of multiple organs and tissues. There has been a significant focus on understanding the role of BMPs in pancreatic ß-cell dysfunction associated with type 2 diabetes (T2D). Our objective was to investigate the relationship between BMP6 and glucose homeostasis. METHODS: Ob/ob mice were treated with BMP6 for 6 days and analyzed for insulin release, body weight, lipid parameters and glucose tolerance. Quantitative real-time PCR, chromatin immunoprecipitation and glucose output assays were used to assess BMP6 effect on gluconeogenesis in rat hepatoma H4IIE cells. Specificity of BMP6 receptors was characterized by the utilization of various receptor Fc fusion proteins in luciferase reporter gene and glucose output assays in INS1 and H4IIE cells. RESULTS: Treatment of ob/ob mice with BMP6 for 6 days resulted in a reduction of circulating glucose and lipid levels, followed by a significantly elevated plasma insulin level in a dose-dependent manner. In addition, BMP6 improved the glucose excursion during an oral glucose tolerance test, lowering the total glycemic response by 21%. In rat H4IIE hepatoma cells, BMP6 inhibited gluconeogenesis and glucose output via downregulation the PepCK expression. Moreover, BMP6 inhibited glucose production regardless of the presence of cAMP, antagonizing its glycogenolytic effect. BMP6 acted on pancreatic and liver cells utilizing Alk3, Alk6 and ActRIIA serine/threonine kinase receptors. CONCLUSIONS: Collectively, we demonstrate that BMP6 improves glycaemia in T2D mice and regulates glucose metabolism in hepatocytes representing an exciting prospect for future treatments of diabetes.

Regulation of muscle protein synthesis in an in vitro cell model using ex vivo human serum.

NEW FINDINGS: What is the central question of this study? Can medium conditioned by ex vivo human serum regulate muscle protein synthesis in skeletal muscle cells in vitro? What is the main finding and its importance? This study demonstrates that medium conditioned by ex vivo human serum can regulate muscle protein synthesis in skeletal muscle cells in vitro via the mammalian Target of Rapomycin (mTOR) pathway, and this can be regulated differentially by fed and fasted ex vivo human serum. ABSTRACT: Human serum embodies the integrated systemic response to any condition or perturbation, which may regulate muscle protein synthesis (MPS). Conditioning of medium with human serum represents a physiologically relevant method of regulating MPS in vitro. The primary purpose of the study was the development of a model using ex vivo human serum to condition medium and regulate MPS in in vitro skeletal muscle cells. Four young healthy men reported to the laboratory after an overnight fast and were fed with 0.33 g (kg body mass)-1 whey protein. Blood samples were taken before (Fasted) and 60 min postprandial (Fed). Fully differentiated C2C12 skeletal muscle cells were nutrient and serum deprived for 1 h and subsequently treated with medium conditioned with Fasted or Fed ex vivo human serum (20%) for 4 h. The MPS was measured using the surface sensing of translation technique and activation of mTOR, P70S6K and 4EBP1 by Western blot. Fasted and fed ex vivo human serum increased MPS (P < 0.05). Although a strong effect (ƞ2  = 0.36) for increased MPS in Fed relative to Fasted was observed, this was not statistically significant (P > 0.05). Activation of mTOR, P70S6K and 4EBP1 was significantly increased after treatment with Fed compared with Fasted ex vivo human serum (P < 0.05). Here, we developed and optimized the conditions for culture of C2C12 skeletal muscle cells, measurement of MPS and signalling in medium conditioned by ex vivo human serum. Furthermore, the functionality of the model was demonstrated by comparison of the response to medium conditioned by Fasted and Fed ex vivo human serum.

Systemic inhibition of BMP1-3 decreases progression of CCl4-induced liver fibrosis in rats.

Liver fibrosis is a progressive pathological process resulting in an accumulation of excess extracellular matrix proteins. We discovered that bone morphogenetic protein 1-3 (BMP1-3), an isoform of the metalloproteinase Bmp1 gene, circulates in the plasma of healthy volunteers and its neutralization decreases the progression of chronic kidney disease in 5/6 nephrectomized rats. Here, we investigated the potential role of BMP1-3 in a chronic liver disease. Rats with carbon tetrachloride (CCl4)-induced liver fibrosis were treated with monoclonal anti-BMP1-3 antibodies. Treatment with anti-BMP1-3 antibodies dose-dependently lowered the amount of collagen type I, downregulated the expression of Tgfb1, Itgb6, Col1a1, and Acta2 and upregulated the expression of Ctgf, Itgb1, and Dcn. Mehanistically, BMP1-3 inhibition decreased the plasma levels of transforming growth factor beta 1(TGFß1) by prevention of its activation and lowered the prodecorin production further suppressing the TGFß1 profibrotic effect. Our results suggest that BMP1-3 inhibitors have significant potential for decreasing the progression of fibrosis in liver cirrhosis.

Exogenous BMP7 corrects plasma iron overload and bone loss in Bmp6-/- mice.

PURPOSE: Iron overload accelerates bone loss in mice lacking the bone morphogenetic protein 6 (Bmp6) gene, which is the key endogenous regulator of hepcidin, iron homeostasis gene. We investigated involvement of other BMPs in preventing haemochromatosis and subsequent osteopenia in Bmp6-/- mice. METHODS: Iron-treated wild-type (WT) and Bmp6-/- mice were analysed for hepcidin messenger RNA (mRNA) and tissue and blood BMP levels by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry, Western blot, enzyme-linked immunosorbent assay (ELISA) and proximity extension assay. BMPs labeled with technetium-99m were used in pharmacokinetic studies. RESULTS: In WT mice, 4 h following iron challenge, liver Bmp6 and hepcidin expression were increased, while expression of other Bmps was not affected. In parallel, we provided the first evidence that BMP6 circulates in WT mice and that iron increased the BMP6 serum level and the specific liver uptake of (99m)Tc-BMP6. In Bmp6-/- mice, iron challenge led to blunted activation of liver Smad signaling and hepcidin expression with a delay of 24 h, associated with increased Bmp5 and Bmp7 expression and increased Bmp2, 4, 5 and 9 expression in the duodenum. Liver Bmp7 expression and increased circulating BMP9 eventually contributed to the late hepcidin response. This was further supported by exogenous BMP7 therapy resulting in an effective hepcidin expression followed by a rapid normalisation of plasma iron values and restored osteopenia in Bmp6-/- mice. CONCLUSION: In Bmp6-/- mice, iron activated endogenous compensatory mechanisms of other BMPs that were not sufficient for preventing hemochromatosis and bone loss. Administration of exogenous BMP7 was effective in correcting the plasma iron level and bone loss, indicating that BMP6 is an essential but not exclusive in vivo regulator of iron homeostasis.

The clinical use of bone morphogenetic proteins revisited: a novel biocompatible carrier device OSTEOGROW for bone healing.

PURPOSE: The purpose of this study was to revise the clinical use of commercial BMP2 (Infuse) and BMP7 (Osigraft) based bone devices and explore the mechanism of action and efficacy of low BMP6 doses in a novel whole blood biocompatible device OSTEOGROW. METHODS: Complications from the clinical use of BMP2 and BMP7 have been systemically reviewed in light of their role in bone remodeling. BMP6 function has been assessed in Bmp6-/- mice by µCT and skeletal histology, and has also been examined in mesenchymal stem cells (MSC), hematopoietic stem cells (HSC) and osteoclasts. Safety and efficacy of OSTEOGROW have been assessed in rats and rabbits. RESULTS: Clinical use issues of BMP2 and BMP7 have been ascribed to the limited understanding of their role in bone remodeling at the time of device development for clinical trials. BMP2 and BMP7 in bone devices significantly promote bone resorption leading to osteolysis at the endosteal surfaces, while in parallel stimulating exuberant bone formation in surrounding tissues. Unbound BMP2 and BMP7 in bone devices precipitate on the bovine collagen and cause inflammation and swelling. OSTEOGROW required small amounts of BMP6, applied in a biocompatible blood coagulum carrier, for stimulating differentiation of MSCs and accelerated healing of critical size bone defects in animals, without bone resorption and inflammation. BMP6 decreased the number of osteoclasts derived from HSC, while BMP2 and BMP7 increased their number. CONCLUSIONS: Current issues and challenges with commercial bone devices may be resolved by using novel BMP6 biocompatible device OSTEOGROW, which will be clinically tested in metaphyseal bone fractures, compartments where BMP2 and BMP7 have not been effective.

Exogenous heparin binds and inhibits bone morphogenetic protein 6 biological activity.

PURPOSE: The purpose of this study was to explore the effect of heparin on bone morphogenetic protein 6 (BMP6) osteogenic activity. METHODS: Western blot analysis was used to confirm the binding of BMP6 to heparin and to observe its effect on BMP6 signaling in C2C12-BRE-Luc myoblasts. Real-time RT-PCR was performed for the expression analysis of alkaline phosphatase (ALP) and osteocalcin (OC) in C2C12 myoblasts treated with BMP6 and heparin for 72 hours. Rat ectopic bone formation assay was performed to explore the effect of heparin on BMP6 osteogenic activity. Two weeks following implantation the implants were analysed morphologically and histologically. A mouse osteoporotic model was used to test the ability of BMP6 to improve the bone quality in vivo in the presence of heparin, followed by DEXA and µCT analyses. Blood coagulation was tested in rats previously treated with BMP6. RESULTS: BMP6 specifically bound to heparin and induced Smad1/5/8 phosphorylation which was inhibited by heparin. After 48 and 72 hours of treatment, heparin inhibited BMP6-induced ALP and OC expression in C2C12 cells. Heparin dose dependently inhibited BMP6-induced new bone and cartilage formation in the rat ectopic bone formation assay, while in osteoporotic mice heparin inhibited the BMP6 potential to improve the bone quality as evidenced by decreased bone mineral density and trabecular bone parameters. Interestingly, BMP6 prevented the effect of heparin on the blood coagulation parameters. CONCLUSION: The interaction of BMP6 with heparin might contribute to the heparin-induced osteoporosis and blood coagulation.